Gordonia cholesterolivorans GN3-13LX and application thereof
By isolating and enriching the Gordonia cholesterolivorans GN3-13LX strain, the problem of insufficient PPC degradation capacity in existing technologies has been solved, achieving efficient biodegradation of PPC with a degradation rate of 8.07%, and the degradation process is safe and efficient.
Patent Information
- Application Number
- CN202511148743.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-15
- Publication Date
- 2025-12-05
AI Technical Summary
There are few strains with PPC degradation capabilities in the existing technology, and the degradation rate of PPC in the natural environment is affected by the type and abundance of microorganisms, making it difficult to degrade efficiently.
A strain of Gordonia cholesterolivorans GN3-13LX is provided, which, by being isolated and enriched from activated sludge in a wastewater treatment plant, is able to survive and degrade petroleum-based biodegradable plastic polypropylene carbonate using PPC as the sole carbon source.
It achieves efficient biodegradation of PPC with a degradation rate of 8.07%, and the degradation process is safe and efficient, the material is easy to obtain, and the preparation efficiency is high.
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Figure CN121065002A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of microorganisms, and in particular to a Gordonia cholesterolivorans GN3-13LX and application thereof. BACKGROUND
[0002] The emergence of plastic products has brought great convenience to human society, but at the same time, due to the improper disposal of a large amount of plastic waste, it has caused serious "white pollution" to the natural environment. With the support of national policy on the use of biodegradable plastics and the enhancement of people's environmental protection consciousness, biodegradable plastics are increasingly concerned. Biodegradable plastics can be degraded under the action of microorganisms under natural conditions (the absolute degradation rate is greater than 60% in 180 days), and finally converted into harmless substances such as water, carbon dioxide and inorganic salts, effectively solving the "white pollution" problem.
[0003] Degradable plastics can be divided into two categories: biobased degradable plastics and petroleum-based degradable plastics. Poly (propylene carbonate) (PPC) belongs to petroleum-based degradable plastics. PPC was first synthesized by Inoue et al. (Inoue S, Koinuma H, Tsuruta T. Copolymerization of carbon dioxide and epoxide with organometallic compounds[J]. Macromol Chem Phys, 1969, 130 (1):210-220.) and Mtibe et al. (Mtibe A, Motloung MP, Bandyopadhyay J, Ray SS. Synthetic Biopolymers and Their Composites: Advantages and Limitations-An Overview[J]. Macromol Rapid Commun, 2021, 42 (15):2100130) in a diethyl zinc / water catalytic system, using carbon dioxide (CO2) and propylene oxide (PO) as raw materials. Subsequently, scholars from various countries have continuously optimized the synthesis process of PPC, achieving technological breakthroughs and industrialization of PPC. Currently, PPC is applied in various fields such as packaging, agriculture, and medical treatment.
[0004] The degradation of PPC in the natural environment is based on hydrolysis, and the species and abundance of microorganisms in the environment of PPC degradation are important factors affecting the degradation rate of PPC. Therefore, finding a microorganism strain that can degrade PPC in the natural environment has become an important research topic for sustainable development. SUMMARY
[0005] The application aims to provide a microbial strain Gordonia cholesterolivorans GN3-13LX and application thereof, the microbial strain Gordonia cholesterolivorans The GN3-13LX strain has strong degradation ability on PPC plastics and can survive by using PPC as the only carbon source.
[0006] In order to achieve the above-mentioned application purposes, the application provides the following technical solutions. The application provides a microbial strain Gordonia cholesterolivorans The GN3-13LX is preserved in the Guangdong Microbial Culture Collection Center on June 4, 2025, and the preservation number is GDMCC No: 66462.
[0007] The application also provides the microbial strain Gordonia cholesterolivorans Application of the GN3-13LX in degrading petroleum-based degradable plastics.
[0008] Preferably, the petroleum-based degradable plastics include polypropylene carbonate.
[0009] The application also provides application of the microbial strain Gordonia cholesterolivorans GN3-13LX in preparing a microbial degrading agent.
[0010] The application also provides a microbial agent, which comprises the microbial strain Gordonia cholesterolivorans GN3-13LX.
[0011] The application also provides application of the microbial agent in degrading petroleum-based degradable plastics.
[0012] Preferably, the petroleum-based degradable plastics include polypropylene carbonate.
[0013] The application also provides application of the microbial agent in preparing a microbial degrading agent.
[0014] The application also provides a method for degrading petroleum-based degradable plastics, which comprises the following steps: The microbial strain Gordonia cholesterolivorans GN3-13LX or the microbial agent is inoculated into a culture medium containing petroleum-based degradable plastics and is cultured.
[0015] Preferably, the petroleum-based degradable plastics include polypropylene carbonate.
[0016] Compared with the prior art, the application has the following beneficial effects: There are few strains having PPC degradation ability, and the application provides the microbial strain Gordonia cholesterolivorans The GN3-13LX has PPC degradation ability, and Gordonia cholesterolivoransGN3-13LX is isolated from activated sludge in a sewage treatment plant, has high use safety, and utilizes Gordonia cholesterolivorans GN3-13LX can realize biodegradation of PPC.
[0017] In the present application Gordonia cholesterolivorans The enrichment and separation method of GN3-13LX is simple, the material used is widely available and easy to obtain, and the preparation efficiency is high. Biological preservation instructions
[0018] The Gordonia cholesterolivorans GN3-13LX has been preserved in Guangdong Microbial Culture Collection Center on June 4, 2025, the preservation address is No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, and the preservation number is GDMCC No: 66462. BRIEF DESCRIPTION OF DRAWINGS
[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.
[0020] Figure 1 For observing GN3-13LX strain by scanning electron microscope Gordonia cholesterolivorans Strain morphology chart when GN3-13LX strain is observed by scanning electron microscope; Figure 2 Scanning electron microscope chart before and after degradation of PPC film; Figure 3 Water contact angle of the film after degradation of PPC film; Figure 4 Result of Fourier infrared spectrum analysis of PPC film. DETAILED DESCRIPTION
[0021] Now a variety of exemplary embodiments of the present application will be described in detail, which should not be considered as a limitation of the present application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present application.
[0022] It should be understood that the terms described in the present application are only for describing the specific embodiments, and are not used to limit the present application. In addition, for the numerical range in the present application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the range and any other stated value or intermediate value in the range is also included in the present application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.
[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0026] This invention provides Gordonia cholesterolivorans GN3-13LX was deposited at the Guangdong Provincial Center for Microbial Culture Collection on June 4, 2025, with accession number GDMCC No: 66462.
[0027] In this invention, the Gordonia cholesterolivorans The GN3-13LX strain exhibits strong degradation capabilities against PPC plastics and can survive using PPC as the sole carbon source. Gordonia cholesterolivorans The preferred method for laboratory preservation of strain GN3-13LX is: Gordonia cholesterolivorans The GN3-13LX strain was inoculated into LB broth medium (10 g / L peptone, 5 g / L sodium chloride, 1 g / L glucose, 5 g / L yeast extract, pH 7.0±0.2). When the OD600 of the strain reached 0.8-1, the bacterial culture was mixed with 50% glycerol preservation solution at a ratio of 1:1 and stored in a -80 ℃ freezer. This preservation method can achieve a shelf life of more than 10 years.
[0028] The present invention also provides the aforementioned Gordonia cholesterolivorans Application of GN3-13LX in the degradation of petroleum-based biodegradable plastics.
[0029] In this invention, the petroleum-based biodegradable plastic is preferably polypropylene carbonate.
[0030] The present invention also provides the aforementioned Gordonia cholesterolivorans Application of GN3-13LX in the preparation of microbial degradation agents.
[0031] The present invention also provides a microbial inoculant, comprising the aforementionedGordonia cholesterolivorans GN3-13LX.
[0032] The present invention also provides the application of the aforementioned microbial agent in the degradation of petroleum-based biodegradable plastics.
[0033] In this invention, the petroleum-based biodegradable plastic is preferably polypropylene carbonate.
[0034] The present invention also provides the application of the aforementioned microbial inoculant in the preparation of microbial degradation agents.
[0035] The present invention also provides a method for degrading petroleum-based biodegradable plastics, comprising the following steps: The above Gordonia cholesterolivorans GN3-13LX or the aforementioned microbial agent is inoculated into a culture medium containing petroleum-based biodegradable plastic for cultivation.
[0036] In this invention, the petroleum-based biodegradable plastic preferably includes polypropylene carbonate; the pre-inoculation preparation preferably involves... Gordonia cholesterolivorans Preliminary culture is performed using GN3-13LX or microbial inoculants, preferably by: Gordonia cholesterolivorans GN3-13LX or microbial inoculants are inoculated into the culture medium and cultured once. Single colonies are then selected and inoculated into liquid culture medium for a second culture to obtain the culture solution. The preferred inoculation method is streak inoculation. The preferred culture medium is LB agar. The preferred temperature for the first culture is 28-32℃, more preferably 30℃. The preferred culture time for the first culture is 1.5-2.5 days, more preferably 2 days. The preferred liquid culture medium is LB liquid culture medium. The preferred temperature for the second culture is 28-32℃, more preferably 30℃. The preferred culture time for the second culture is 20-25 hours, more preferably 22-25 hours. The culture time is preferably 4 hours, more preferably 23 hours; the rotation speed of the secondary culture is preferably 140~160 r / min, more preferably 145~155 r / min, and even more preferably 150 r / min; the culture medium containing petroleum-based biodegradable plastic is preferably MSM inorganic salt liquid culture medium; the culture temperature is preferably 28~32℃, more preferably 30℃; the culture time is preferably 28~32 days, more preferably 30 days; the rotation speed of the culture is preferably 140~160 r / min, more preferably 145~155 r / min, and even more preferably 150 r / min.
[0037] Example 1 Gordonia cholesterolivorans Enrichment and isolation of GN3-13LX strain Activated sludge was collected from a sewage treatment plant in Huizhou, 23°13'28" N, 114°18'54" E. The activated sludge was mixed with a sterile trace carbon source liquid medium (Yeast extract 0.5 g / L, (NH4)2SO4 2 g / L, FeSO4·7H2O 1 g / L, MgSO4·7H2O 1 g / L, CuSO4·5H2O 0.1 g / L, MnSO4·H2O 0.1 g / L, ZnSO4·7H2O 0.1 g / L) by shaking at a volume ratio of 1:10. 100 mg of sterilized PPC membrane was added, and the mixture was enriched at 30°C and 150 r / min for 30 days. The enriched PPC membrane was transferred to a 15-ml centrifuge tube containing 10 ml of normal saline, and then the bacterial suspension was obtained by shaking and centrifugation.
[0038] 1 ml of the bacterial suspension was added to a test tube containing 9 ml of sterile water, and mixed by repeatedly blowing with a pipette. This was 10 -1 dilution. -1 1 ml of the 10 -2 dilution was transferred to a new test tube, 9 ml of sterile water was added, and the mixture was repeatedly blown to obtain 10 -4 dilution. In this way, 10 -5 , 10 -6 dilutions were obtained.
[0039] 50 μl of each 10 -4 dilution was taken with a pipette and spread on 3 LB agar culture media (purchased from HKM, product number 028330) using a sterilized and cooled spreader. In the same way, 10 -5 , 10 -6 dilutions were obtained. The culture media were incubated at 30°C until the colonies grew.
[0040] A single colony was taken with a sterilized and cooled inoculation loop and streaked onto LB agar culture medium, which was incubated at 30°C for 2 days. A single colony was again taken with a sterilized and cooled inoculation loop and streaked for culture, until a pure culture was obtained. The purified strain was inoculated into sterile LB broth culture medium, which was incubated at 30°C and 150 rpm for 24 h, and then preserved in glycerol at -80°C.
[0041] The separated PPC-degrading bacteria are verified for their PPC-degrading ability one by one in MSM inorganic salt liquid medium (NH4Cl 1 g / L, MgSO4·7H2O 0.2 g / L, K2HPO4 1 g / L, CaCl2·2H2O 0.1 g / L, KCl 0.15 g / L, FeSO4·6H2O 0.001 g / L, ZnSO4·7H2O 0.001 g / L, MnSO4 0.001 g / L, pH 7.8) with PPC as the sole carbon source. Specifically, the liquid medium is treated by sterilization at 121°C for 20 min, about 100 mg of PPC film is put into the liquid medium, the above separated microorganisms are inoculated, and the culture is incubated at 30°C and 150 rpm in a constant-temperature incubator for 30 days. The bacteria that can grow with PPC as the sole carbon source are screened out.
[0042] Example 2 Identification of PPC-degrading bacterial strains 2.1 Under observation by a scanning electron microscope, rod-shaped bacteria can be obviously observed, and obvious annular protuberance structures can be observed on the rod-shaped bacteria. As shown in FIG. 1. Figure 1
[0043] 2.2 16S r DNA sequence determination: The 16S r DNA sequence of the separated strain is determined, and the obtained sequence result is subjected to BLAST search comparison in the National Center for Biotechnology Information (NCBI).
[0044] The comparison result shows that the sequence of the strain GN3-13LX has the highest similarity with Chol-3T, which is 99.79%, so that the separated strain is determined to be Gordonia cholesteroliphila (Gordonia cholesteroliphila), and is named as GN3-13LX. Gordonia cholesterolivorans Gordonia cholesterolivorans
[0045] The strain GN3-13LX is a strain of Gordonia cholesteroliphila, and has the following advantages: Gordonia cholesterolivorans
[0046] Example 3 Gordonia cholesterolivorans Test of degradation ability of GN3-13LX to PPC The GN3-13LX strain was inoculated into LB agar medium by streak inoculation method, and cultured at 30°C for 2d. The single colony of the GN3-13LX strain was inoculated into LB broth medium (purchased from HKM, item number 028320), and cultured at 30°C, 150rpm for 24h. The above culture liquid was inoculated into 80ml MSM inorganic salt liquid medium containing 50mg PPC at an inoculation amount of 2% (volume fraction), and cultured at 30°C, 150r / min for 30d. The degradation of PPC was detected every 10d. Gordonia cholesterolivorans The GN3-13LX strain was inoculated into LB agar medium by streak inoculation method, and cultured at 30°C for 2d. The single colony of the GN3-13LX strain was inoculated into LB broth medium (purchased from HKM, item number 028320), and cultured at 30°C, 150rpm for 24h. The above culture liquid was inoculated into 80ml MSM inorganic salt liquid medium containing 50mg PPC at an inoculation amount of 2% (volume fraction), and cultured at 30°C, 150r / min for 30d. The degradation of PPC was detected every 10d. Gordonia cholesterolivorans The GN3-13LX strain was inoculated into LB agar medium by streak inoculation method, and cultured at 30°C for 2d. The single colony of the GN3-13LX strain was inoculated into LB broth medium (purchased from HKM, item number 028320), and cultured at 30°C, 150rpm for 24h. The above culture liquid was inoculated into 80ml MSM inorganic salt liquid medium containing 50mg PPC at an inoculation amount of 2% (volume fraction), and cultured at 30°C, 150r / min for 30d. The degradation of PPC was detected every 10d.
[0047] The results showed that: Gordonia cholesterolivorans The GN3-13LX strain had obvious degradation to PPC, and the average weight loss of PPC reached 8.07% in 30d.
[0048] Example 4 Microscope observation The sterile treatment (121°C sterilization for 20min, CK) and the GN3-13LX treated PPC film in Example 3 were taken out from the culture medium, and the film surface was gently washed. The film was dried and fixed for gold spraying treatment. The scanning size was 5mm×5mm, and the film was observed under a scanning electron microscope. The results are shown in Gordonia cholesterolivorans The GN3-13LX treated PPC film was taken out from the culture medium, and the film surface was gently washed. The film was dried and fixed for gold spraying treatment. The scanning size was 5mm×5mm, and the film was observed under a scanning electron microscope. The results are shown in Figure 2
[0049] The results showed that the surface of the PPC film appeared wrinkles and holes and other damage.
[0050] Example 5 Measurement of contact angle The contact angle was measured by controlled hanging drop method, and the hydrophilicity of the sterile treatment (121°C sterilization for 20min) and the GN3-13LX treated PPC sample in Example 3 were analyzed. Specifically as follows: Gordonia cholesterolivorans The contact angle was measured by controlled hanging drop method, and the hydrophilicity of the sterile treatment (121°C sterilization for 20min) and the GN3-13LX treated PPC sample in Example 3 were analyzed. Specifically as follows: 2μl of deionized water was dropped on the surface of the PPC material, and a camera with a magnifying glass was used to take pictures immediately. The water contact angle readings were calculated by recording multiple times. The results are shown in Figure 3 The contact angle was measured by controlled hanging drop method, and the hydrophilicity of the sterile treatment (121°C sterilization for 20min) and the GN3-13LX treated PPC sample in Example 3 were analyzed. Specifically as follows: The contact angle was measured by controlled hanging drop method, and the hydrophilicity of the sterile treatment (121°C sterilization for 20min) and the GN3-13LX treated PPC sample in Example 3 were analyzed. Specifically as follows:
[0051] The results showed that the water contact angle of the sterile treatment was 87.90°±1.90°, Gordonia The results showed that the water contact angle of the sterile treatment was 87.90°±1.90°, cholesterolivorans The results showed that the water contact angle of the sterile treatment was 87.90°±1.90°, Gordonia The results showed that the water contact angle of the sterile treatment was 87.90°±1.90°, cholesterolivoransThe PPC water contact angle treated with GN3-13LX was significantly smaller than that of the sterile treatment, indicating that... Gordonia cholesterolivorans The hydrophobicity of the PPC membrane surface decreased after GN3-13LX treatment.
[0052] Example 6: Fourier Transform Infrared Spectroscopy Detection Fourier transform infrared spectroscopy was used to measure the infrared spectrometer at 4000-400 cm⁻¹. -1 The analysis was conducted within the range of aseptic treatment (sterilization at 121°C for 20 min) and in Example 3. Gordonia cholesterolivorans Structural changes in PPC after GN3-13LX treatment. For example... Figure 4 As shown.
[0053] FTIR spectroscopy shows that the PPC film... Gordonia cholesterolivorans After 30 days of degradation, GN3-13LX was at 2921 cm⁻¹ -1 1756cm -1 1713cm -1 1453cm -1 1268cm -1 1181cm -1 1083cm -1 and 728cm -1 The vibration peak at that location was significantly weakened.
[0054] As can be seen from the above embodiments, the present invention provides a Gordonia cholesterolivorans GN3-13LX and its applications, the Gordonia cholesterolivorans GN3-13LX is separated from activated sludge from wastewater treatment plants and has a high degree of safety in use. Gordonia cholesterolivorans Gordonia cholesterolivorans GN3-13LX enables the biodegradation of PPC.
[0055] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A kind Gordonia cholesterolivorans GN3-13LX, characterized in that, Gordonia cholesterolivorans deposited on June 4, 2025 in the Guangdong Provincial Microbial Culture Collection, with the accession number GDMCC No: 66462.
2. The method of claim 1, wherein the method is performed in vivo. Gordonia cholesterolivorans Use of GN3-13LX in degrading petroleum-based degradable plastics.
3. Use according to claim 2, characterized in that, The petroleum-based degradable plastics include polypropylene carbonate.
4. The method of claim 1, wherein the method is performed in vivo. Gordonia cholesterolivorans Use of GN3-13LX in the preparation of a microbial degradation agent.
5. A microbial inoculant, characterized in that, including the method of claim 1 Gordonia cholesterolivorans GN3-13LX.
6. The microbial agent of claim 5 in the degradation of petroleum-based degradable plastics.
7. Use according to claim 6, characterized in that, The petroleum-based degradable plastics include polypropylene carbonate.
8. The microbial agent of claim 5 in the preparation of a microbial degrading agent.
9. A method of degrading a petroleum-based degradable plastic, characterized by, comprising the following steps: The microorganism inoculum of claim 1 Gordonia cholesterolivorans GN3-13LX or the microbial inoculum of claim 5 is inoculated into a culture medium containing petroleum-based degradable plastics and cultured.
10. The method of claim 9, wherein, The petroleum-based degradable plastics include polypropylene carbonate.